1. Field of the Invention
The inventive subject matter relates to a method of inducing an immune response against enterotoxigenic Escherichia coli using bacterial fimbrial components. The method contemplates using enterotoxigenic Escherichia coli major and minor fimbrial subunits, incorporated into a stabilizing construct, as immunogens.
2. Description of Related Art
Enterotoxigenic Escherichia coli (ETEC) are a principal cause of diarrhea in young children in resource-limited countries and in travelers to these areas (Black, R. E. Rev. Infect. Dis. 12 (Suppl. 1): S73-79 (1990); Huilan, et al., Bull. World Health Organ. 69: 549-55 (1991)). ETEC-associated diarrheal disease is mediated by bacterial adherence to small intestinal epithelial cells and expression of a heat-labile (LT) and/or heat-stable (ST) enterotoxin (Nataro and Kaper, Clin. Microbiol. Rev. 11: 142-201 (1998)). ETEC typically attach to host cells via filamentous bacterial surface structures known as colonization factors (CFs). More than 20 different CFs have been described, a minority of which have been unequivocally incriminated in pathogenesis (Gaastra and Svennerholm, Trends Microbiol., 4: 444-452 (1996)).
Evidence for a pathogenic role exists for colonization factor antigen I (CFA/I), the first human-specific ETEC CF to be described. CFA/I is the archetype of a family of eight ETEC fimbriae that share genetic and biochemical features (Evans, et al., Infect. Immun., 12: 656-667 (1975); Gaastr and Svennerholm, Trends Microbiol., 4: 444-452 (1996); Grewal, et al., Infect. Immun., 65: 507-513 (1997)). This family includes coli surface antigen 1 (CS1), CS2, CS4, CS14, CS17, CS19 and putative colonization factor 071 (PCFO71). The complete DNA sequences of the gene clusters encoding CFA/I, CS1 and CS2 have been published (Froehlich, et al., Mol. Microbiol., 12: 387-401 (1994); Froehlich, et al., Infect. Immun., 63: 4849-56 (1995); Perez-Casal, et al., Infect. Immun. 58: 3594-3600 (1990); Scott, et al., Mol. Microbiol. 6: 293-300 (1992); Anantha, et al., Inf. And Imm., 72: 7190-7201 (2004)). The genes for the major subunit of two of the other related fimbriae have also been reported (Gaastra, et al., Int. J. Med. Microbiol. 292: 43-50 (2002); Grewal, et al., Infect. Immun. 65: 507-513 (1997). The four-gene bioassembly operons of CFA/I, CS1, and CS2 are similarly organized, encoding (in order) a periplasmic chaperone, major fimbrial subunit, outer membrane usher protein, and minor fimbrial subunit. CFA/I assembly takes place through the alternate chaperone pathway, distinct from the classic chaperone-usher pathway of type I fimbrial formation and that of other filamentous structures such as type IV pili (Ramer, et al., J. Bacteriol., 184: 3457-65 (2002); Soto and Hultgren., J. Bacteriol., 181: 1059-1071 (1999). Based on the primary sequence of the major fimbrial subunit, CFA/I and related fimbriae have been grouped as class 5 fimbriae.
Distinct from class 5 fimbriae, coli surface antigen 3 (CS3) represents the common adhesive fibrilla of the ETEC colonization factor antigen II (CFA/II) complex. ETEC expressing these antigens are prevalent in many parts of the world. Although the conformational nature of CS3 containing fibrillae are less understood than class 5 fimbriae, it is anticipated that these structures are important for eliciting anti-ETEC immune protection. Similarly, coli surface antigen 6 (CS6) (Tobias, et al., Vaccine., 26: 5373-5380 (2008)) has also been described and associated with ETEC mediated diarrheal disease (Gaastra and Svennerholm., Trends Microbiol., 4: 444-452 (1996); Qadri, et al., Clin. Microbiol., Rev. 18: 465-483 (2005); Sack, et al., Vaccine, 25: 4392-4400 (2007); Al-Gallas, et al., Am. J. Trop. Med. Hyg. 77: 571-582 (2007)).
Studies of CS1 have yielded details on the composition and functional features of Class 5 fimbriae Sakellaris and Scott, Mol. Microbiol. 30: 681-687 (1998). The CS1 fimbrial stalk consists of repeating CooA major subunits. The CooD minor subunit is allegedly localized to the fimbrial tip, comprises an extremely small proportion of the fimbrial mass, and is required for initiation of fimbrial formation (Sakellaris, et al., J. Bacteriol., 181: 1694-1697 (1999). Contrary to earlier evidence suggesting that the major subunit mediates binding (Buhler, et al., Infect. Immun. 59: 3876-3882 (1991), findings have implicated the minor subunit as the adhesin and identified specific amino acid residues required for in vitro adhesion of CS1 and CFA/I fimbriae Sakellaris, et al., PNAS (USA) 96: 12828-12832 (1999). The inferred primary amino acid structure of those major subunits that have been sequenced share extensive similarity. Serologic cross-reactivity of native fimbriae is, however, limited, and the pattern of cross-reactivity correlates with phylogenetically defined subtaxons of the major subunits (Gaastra, et al., Int. J. Med. Microbiol., 292: 43-50 (2002).
Studies to examine the evolutionary relationships of the minor and major subunits of Class 5 ETEC fimbriae as well as the two assembly proteins have been conducted (Anantha, et al., Inf. Imm., 72: 7190-7201 (2004)). The results demonstrated that evolutionary distinctions exist between the Class 5 major and minor fimbrial subunits and that the minor subunits function as adhesins.
The major subunit alleles of CS4, CS14, CS17 and CS19 gene clusters each showed 99-100% nucleotide sequence identity with corresponding gene sequence(s) previously deposited in GenBank, with no more than four nucleotide differences per allele. Each locus has four open reading frames that encoded proteins with homology to the CFA/I class chaperones, major subunits, ushers and minor subunits. As previously reported Gaastra, et al., Int. J. Med. Microbiol., 292: 43-50 (2002), the one exception was for the CS14 gene cluster, which contained two tandem open reading frames downstream of the chaperone gene. Their predicted protein sequences share 94% amino acid identity with one another and are both homologous to other Class 5 fimbriae major subunits.
Examination of the inferred amino acid sequences of all the protein homologs involved in Class 5 fimbrial biogenesis reveals many basic similarities. Across genera, each set of homologs generally share similar physicochemical properties in terms of polypeptide length, mass, and theoretical isoelectric point. All of the involved proteins contain an amino-terminal signal peptide that facilitates translocation to the periplasm via the type II secretion pathway. None of the major subunit proteins contain any cysteine residues, while the number and location of six cysteine residues are conserved for all of the minor subunits except that of the Y. pestis homolog 3802, which contains only four of these six residues.
Type 1 and P fimbriae have been useful models in elucidating the genetic and structural details of fimbriae assembled by the classical chaperone-usher pathway (23, 24, 25). An outcome of work with type 1 and P fimbriae (Kuehn, et al., Nature, 356: 252-255 (1992); Sauer, et al., Science, 285: 1058-1061 (1999); Choudhury, et al., Science, 285: 1061-1066 (1999)) has led to the development of the principle of donor strand complementation, a process in which fimbrial subunits non-covalently interlock with adjoining subunits by iterative intersubunit sharing of a critical, missing β-strand (Barnhart, et al, PNAS (USA), 97: 7709-7714 (2000); Viboud, et al., Microb. Athogen, 21: 139-147 (1996)).
The eight ETEC Class 5 fimbriae clustered into three subclasses of three (CFA/I, CS4, and CS14), four (CS1, PCFO71, CS17 and CS19), and one (CS2) member(s) (referred to as subclasses 5a, 5b, and 5c, respectively) (21). Previous reports demonstrated that ETEC bearing CFA/I, CS2, CS4, CS14 and CS19 manifest adherence to cultured Caco-2 cells (6, 22). However, conflicting data have been published regarding which of the component subunits of CFA/I and CS1 mediate adherence (19, 20).